[unreadable] [unreadable] Nonalcoholic steatohepatitis (NASH) associated with obesity and type II diabetes is emerging as one of the most common etiologies of chronic liver disease. Recent evidence supports a "two-hit" hypothesis for NASH with the "first-hit" involving the accumulation of fat in hepatocytes as a consequence of insulin resistance followed by the "second-hit", which entails mitochondrial dysfunction and oxidative and nitrosative stress. Thus, the mitochondrion is postulated to play a central role in the development of NASH with disrupted energy metabolism and excess oxidant production contributing to pathology. The mechanism by which oxidative stress leads to mitochondrial damage is likely to involve the post-translational modification of mitochondrial proteins. Oxidative modifications to mitochondrial proteins will impair mitochondrial bioenergetics and lead to further increases in oxidants, which are proposed to play a key role in the progression from simple fatty liver to NASH. The molecular pathways and targets that underlie these changes to mitochondria in NASH are unknown and the focus of this application. One factor that has hindered progress in elucidating the molecular mechanisms of NASH and development of targeted therapeutics is the lack of appropriate experimental animal models. Preliminary data indicate that feeding a high-fat diet to mice reproduces the key histopathologic features of NASH with mitochondrial dysfunction. Based on these observations it is hypothesized that the inability to adapt to the metabolic alterations associated with obesity results in the overproduction of mitochondrial oxidants, which leads to post-translational modification of proteins, impaired mitochondrial function, and NASH. The first aim of this exploratory project is to characterize and quantify the development of NASH and mitochondrial damage in response to a high-fat diet in mice. The use of this model will facilitate future studies with transgenic and/or knockout mouse models to investigate mechanisms of protection from metabolic stress in liver mitochondria. The second aim is to characterize post-translational modifications to the hepatic mitochondria proteome in response to NASH. Completion of these studies using novel proteomic approaches will enable the identification of the molecular defects in metabolic pathways and/or proteins in NASH that can be targeted by mitochondria-specific therapeutic strategies for treatment and prevention. Public health relevance (lay description) - Studies report an increasing prevalence of obesity in the world. Obesity is associated with serious health problems including heart disease and diabetes and is recognized as the most common risk factor for development of chronic non alcohol-related liver diseases. The studies in this application are important as they will identify the underlying factors responsible for obesity-related liver diseases, which will lead to the discovery of new treatments. [unreadable] [unreadable] [unreadable] [unreadable] [unreadable]